1. Field of the Invention
This invention relates to devices and methods for the generation of ions of heavy molecules, especially biomolecules, in mass spectrometers by bombarding them with uncharged clusters of molecules.
2. Description of the Related Art
In the document EP 1 200 984 B1 (C. Gebhardt and H. Schroder, 1999; corresponding to U.S. Pat. No. 7,247,845 B1), an ionization of large analyte molecules located on the surface of a solid sample support by bombardment with uncharged clusters of molecules is described. The clusters are generated from polar molecules, such as H2O or SO2, within a supersonic jet. The document also discusses the literature in detail, which predominantly investigates the ionization of cluster fragments which are generated by the impact of electrically charged and electrically accelerated clusters on surfaces, but not the ionization of large analyte molecules on sample supports by uncharged clusters.
The term “cluster” usually refers to ensembles of atoms or molecules which are relatively weakly bound by physical forces such as van-der-Waals forces or hydrogen bridge bonds, for example, and whose density is comparable with that of liquids or solids, but which outwardly have the character of a gas phase particle. The cluster size can be adjusted to suit the application and can range from a few tens to many thousands or even hundred thousands of molecules. Correspondingly, the clusters have diameters which range from one to a hundred nanometers.
The clusters can be generated from gaseous cluster substance molecules in many different ways. In a particularly simple method, which was first described in the document referenced above, the cluster substance molecules are added to a carrier gas, such as helium, at concentrations of one to three percent, sometimes up to five percent. The carrier gas is allowed to expand through a suitable nozzle connected to a switching valve in short pulses of 50 to 200 microseconds duration from a pressure of 1×106 to 2×106 pascal (10-20 bar) into a good vacuum of better than 10−3 pascal, such as 10−1 pascal or even less. The adiabatic expansion produces a cold supersonic gas jet, and the condensation of the cluster substance molecules into uncharged clusters takes place within the nozzle and in a short segment behind it. Quantity and size of the clusters are determined by the starting pressure, the starting temperature, the concentration of the cluster substance molecules and by the diameter and shape of the switching valve nozzle. They also depend on the type of carrier gas used. Clusters generated in this way have a broad size distribution. Since the cluster substance molecules introduce the binding energy for each molecule into the cluster as thermal energy during condensation, and since cooling in the light carrier gas is very slow, such a cluster resembles a liquid or solid particle that is always in equilibrium between vaporization and condensation of cluster substance molecules, just below the boiling point at the corresponding pressure within the supersonic jet. This makes the cluster extremely unstable. When the carrier gas is hydrogen, the clusters formed are smaller than those formed in helium because the lower mass of hydrogen means even less cooling of the cluster is available for cluster growth in hydrogen than in helium.
In this generation method, the clusters are also simultaneously accelerated to the velocity of the supersonic gas jet and can be fired onto the sample support plate with this velocity. This velocity can be controlled. Depending on the type of gas and the starting temperature, such a supersonic gas jet can reach velocities of the order of 1,500 to 2,000 meters per second for (pure) helium, and even 2,500 to 3,500 meters per second for (pure) hydrogen. The addition of the heavier cluster substance molecules reduces these velocities accordingly by some 10 to about 30 percent for the gas molecules as well as for the clusters. These velocities are still so high that the kinetic energy Ekin per cluster substance molecule is comparable to the average binding energy Ebind of the molecules in the cluster group. Depending on the ratio of Ekin and Ebind, the cluster can therefore be more or less completely decomposed into the individual cluster substance molecules when impacting onto the surface of a solid body, i.e., the cluster is transformed into a hot gas with high pressure.
The cluster substance molecules which have been investigated most are SO2 and H2O, but the substance HNO3 (nitric acid), which dissociates easily, has also been applied.
The bombardment of the sample support plate with clusters requires a good vacuum. In an environment of 10−3 pascal, the unstable clusters fly to the sample support plate without being destroyed. However, the pressure increases rapidly due to the inflowing gas of the supersonic gas jet and, depending on the pumping capacity and quantity of inflowing gas, reaches pressures above 10 pascal in 10 to 1,000 microseconds. At these pressures, the clusters already noticeably decompose. At pressures of 100 pascal, for example, the clusters are completely decomposed after a short distance of a few centimeters. The path of the supersonic gas jet from the nozzle to the sample support plate must therefore be maintained at a pressure below ten pascal, preferably below one pascal, at least for the desired duration of the bombardment. Since the pumping capacity is variable only to a small extent, the pressure increase essentially depends on the diameter of the nozzle, which determines the inflowing quantity of gas. The pressure of around 10−3 pascal is to be restored by the time of the next supersonic gas pulse; this limits the supersonic pulses to a rate of around 10 to 20 pulses per second; sometimes, however, also up to 100 pulses per second.
As stated above, the clusters have diameters of far less than one micrometer, for example, on a nanometer scale. At a velocity of 1,000 meters per second, the impact takes less than one picosecond from the first contact until standstill. The kinetic energy of the cluster is converted into thermal energy. Even before impact, the clusters already form unstable particles just below boiling point. So when an impact occurs, a compressed gas cloud of free cluster substance molecules with the density of a liquid, and therefore a very high pressure (possibly one thousand bar or higher) and a very high kinetic temperature (possibly one thousand kelvin or higher), is formed due to an immediate phase transition from solid or liquid to gaseous. The question is still unanswered as to whether a large proportion of the cluster substance molecules is ionized, like in a plasma, because the time to assume an equilibrium ionization according to the Saha-Eggert equation may not be available. Fast chemical reactions can, however, occur in the expanding gas cloud, such as a reaction of SO2 and water, which was adsorbed on the sample support plate, to form H2SO3, so proton donors are available in the gas cloud. In the short time of less than one picosecond, the cluster is flattened, and the crushing of the molecules on the sample support plate entrains water and analyte molecules and embeds them into the gas cloud. Large biochemical analyte molecules are often already present in ionized form on the sample support and surrounded by water solvate sheaths, so they are transferred into the gas cloud as ions. As the analyte molecules and analyte ions are embedded into the hot gas of the gas cloud, their internal energy is hardly increased because the process of energy absorption into the inside of the molecule takes considerably longer.
The hot gas cloud now expands adiabatically into the surrounding vacuum, thus reducing the kinetic temperature very quickly, and supersonic speeds are again reached in the front of the gas cloud. During this adiabatic expansion, cluster substance molecules can also condense again as micro-clusters. On the picosecond scale the adiabatic expansion proceeds very slowly, however. During the first picoseconds the inertia of the molecules means that the gas cloud expands by only a few nanometers; only after a million picoseconds, i.e. a microsecond, has the gas cloud expanded to a diameter of around half a millimeter. Velocities of the order of 500 to 1,000 meters per second are attained here in the front of the gas cloud; in the back portion of the gas cloud, near the sample support plate, the velocities are much lower. The local velocities of the cluster substance molecules in the gas cloud are approximately proportional to their distance from the sample support plate.
The adsorbed analyte molecules and analyte ions, which are quite unavoidably taken up in this process, are found in the cloud near to the sample support plate and are therefore accelerated to lower velocities in the range from close to zero up to 100 meters per second. Nevertheless, since their mass is often large, they achieve kinetic energies which have a very broad distribution in the range from close to zero right up to 100 electronvolts and more.
Water molecules adsorbed on the sample support or on the analyte molecules combine with different suitable cluster substance molecules to form proton donors, which are available for proton transfers. When SO2 is used as the cluster substance, for example, H2SO3 is formed with the water molecules, dissociated to a high degree into free protons and SO32−. Even if not dissociated, H2SO3 easily releases protons if a substance with proton affinity is nearby. If the uncharged analyte molecules have surface regions which have a proton affinity, they can accept free protons or protons by proton transfer if they are in the close vicinity, and thus form further analyte ions. The analyte molecules which were already charged when stationary, or are first ionized in the gas, can now be extracted by electric fields and fed to an application. However, it is highly disadvantageous here that their kinetic energies have a very broad distribution, since this makes good ion-optical focusing impossible.
The document EP 1 200 984 B1 referenced above already disclosed that an ionization of analyte molecules by cluster bombardment could be used in mass spectrometric ion sources, for example in ion sources for time-of-flight mass spectrometers, but the document does not provide details of the technique to be used to capture the analyte ions with a high yield. The generation and capture of the analyte ions should produce a high yield in such ion sources, but even the capture of the analyte ions presents a technical problem.
In view of the above, there is a need for devices and methods to produce analyte ions from analyte molecules on sample support plates by cluster bombardment with a high yield and to capture them in such a way that they can be introduced into a mass spectrometer with a high efficiency.